title: Superoxide constitutes a major signal of mitochondrial superoxide flash.
abstract: OBJECTIVE
  Mitochondrial flashes detected with an N- and C-terminal circularly-permuted yellow fluorescent protein (cpYFP) have been thought to represent transient and quantal bursts of superoxide production under physiological, stressful and pathophysiological conditions. However, the superoxide nature of the cpYFP-flash has been challenged, considering the pH-sensitivity of cpYFP and the distinctive regulation of the flash versus the basal production of mitochondrial reactive oxygen species (ROS). Thus, the aim of the study is to further determine the origin of mitochondrial flashes.
  
  
  METHODS
  We investigated the origin of the flashes using the widely-used pH-insensitive ROS indicators, mitoSOX, an indicator for superoxide, and 2, 7-dichlorodihydrofluorescein diacetate (DCF), an indicator for H2O2 and other oxidants.
  
  
  RESULTS
  Robust, quantal, and stochastic mitochondrial flashes were detected with either mitoSOX or DCF in several cell-types and in mitochondria isolated from the heart. Both mitoSOX-flashes and DCF-flashes showed similar incidence and kinetics to those of cpYFP-flashes, and were equally sensitive to mitochondria-targeted antioxidants. Furthermore, they were markedly decreased by inhibitors or an uncoupler of the mitochondrial electron transport chain, as is the case with cpYFP-flashes. The involvement of the mitochondrial permeability transition pore in DCF-flashes was evidenced by the coincidental loss of mitochondrial membrane potential and matrix-enriched rhod-2, as well as by their sensitivity to cyclosporine A.
  
  
  CONCLUSIONS
  These data indicate that all the three types of mitochondrial flashes stem from the common physiological process of bursting superoxide and ensuing H2O2 production in the matrix of single mitochondrion.
authors.affiliation: Department of Physiology, The Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhanglong
authors.initials: Z
authors.lastname: Huang
authors.forename: Tingting
authors.initials: T
authors.lastname: Hou
authors.forename: Jiejia
authors.initials: J
authors.lastname: Xu
authors.forename: Yanru
authors.initials: Y
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Shang
authors.forename: Tao
authors.initials: T
authors.lastname: Ye
authors.forename: Heping
authors.initials: H
authors.lastname: Cheng
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Xianhua
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008929:Mitochondria, Heart / Q000378:metabolism*; D013269:Stochastic Processes; D013481:Superoxides / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D013481:Superoxides; D006861:Hydrogen Peroxide
keywords: Mitochondria; ROS indicators; Reactive oxygen species